1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for the identification and selection of insertional mutants.
2. Description of Related Art
Mutants are powerful tools in the investigation of physiological, developmental, and cell biological processes. Starting with a phenotypic mutant generated by chemical mutagenesis, it is possible to use a genetic map-based strategy to clone a gene (Arondel et al., 1992). Mutations derived from insertional mutagenesis are particularly useful in that they provide xe2x80x9ctaggedxe2x80x9d copies of the mutated gene which may readily be cloned (Yanofsky et al., 1990). However, molecular genetic techniques have advanced such that today most genes are cloned and sequenced long before their function is characterized genetically (Newman et al., 1994). For many genes, phenotypic screens are not available, and mutations which cause lethality remain undetectable. What has been missing is a simple and reliable strategy to go from a gene or protein sequence to the identification of specific mutants.
One solution to problems associated with mutant identification was to use the polymerase chain reaction (PCR) to screen for P-element mutations in sequenced genes of Drosophila (Ballinger et al., 1989; Kaiser et al., 1990). This approach also enhanced the genetics of Caenorhabditis (Rushforth et al., 1993; Zwaal et al., 1993), where transposable element mutations are now commonly isolated for known gene sequences. In these systems, transposon-induced mutations are isolated for known gene sequences by the general strategy known as xe2x80x9csite-selectedxe2x80x9d mutagenesis. Basically, the method relies on the power of PCR to amplify a collection of specific junction fragments between an inserted element and a known target gene sequence from large pools of randomly inserted elements. One primer is used which is homologous to the end of the inserted element with its 3xe2x80x2 end facing outward and one primer within the target gene is used to amplify the sequences at the junction of the insertion. In plants, similar approaches have been used to identify insertion mutations in Petunia, using the transposon dTph1 (Koes et al. 1995), and in Arabidopsis using collections of T-DNA transformed lines (Krysan et al., 1996; Mckinney et al., 1995). In Krysan et al. (1996), 9100 independent T-DNA-transformed Arabidopsis lines (averaging 1.4 insertions per genome) were subjected to site-selected mutagenesis and 17 T-DNA insertions within 63 genes were identified.
While techniques based on the gene-specific amplification of insertional junctions have been useful in the isolation of a number of mutants, they have had limited success in applications toward large-scale genomic investigations. The need for individual amplifications of each gene being investigated represents a significant hindrance when seeking to identify more than a small number of insertional mutants. There is, therefore, a great need in the art for a method by which large numbers of insertional mutants may be rapidly and efficiently identified.
The present invention seeks to overcome deficiencies in the prior art by providing a highly efficient method for selecting insertion events. Therefore, one apsect of the current invention is a method for identifying an insertion event in a genome comprising the steps of: (a) preparing a first DNA composition enhanced for a plurality of insertion junctions; (b) preparing at least a first detectable array including the first DNA composition; and (c) detecting the insertion event from the first array. The step of preparing a first DNA composition may comprise amplification of insertion junctions with inverse PCR, vectorette PCR, primer-adapted PCR, AIMS or any other suitable procedure. The method can further comprise preparing at least a second DNA composition, and additionally any greater number of DNA compositions desired by the user of the invention. The additional DNA compositions may be prepared on the same, or other arrays, as desired by the user of the invention.
In another aspect of the invention, the detectable array can comprise the first and second DNA compositions arranged on a solid support. The solid support can be a microscope slide, and the insertion event can be detected by hybridization with a fluorescently labeled probe comprising cloned DNA, and/or be detected by hybridization with a probe labeled with an antigen, where the antigen is detected with a molecule which binds the antigen. Alternatively, the insertion event can be detected by PCR. In another embodiment of the invention, the array has a solid support comprising a nitrocellulose filter, and the insertion event can be detected by hybridization with a radioactively-labeled probe comprising cloned DNA. The method of detecting can further comprise hybridization of a gene-specific probe to the array. In particular embodiments, the DNA compositions of the array will comprise DNA which has been pooled from multiple individuals. The DNA in the compositions can be derived from potentially any species, including DNA from plants, animals, prokaryotes and lower eukaryotes. In particular embodiments, the DNA may be from a monocot plant, and may further defined as from maize, rice, wheat, barley, sorghum, oat, or sugarcane. In other embodiments, the monocot DNA is maize DNA. The plant DNA may also be dicot DNA, and may be derived from a species selected from the group consisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. In particular embodiments of the invention the dicot plant DNA is Arabidopsis thaliana DNA. In still other embodiments the DNA is animal DNA.
Still yet another aspect of the invention provides a method of determining the function of a DNA sequence. In particular embodiments of the invention the method comprises the steps of: (a) amplifying a plurality of insertion junctions from a DNA composition comprising insertion mutations; (b) creating at least a first array comprising said insertion junctions; (c) detecting at least a first mutation in said DNA sequence from said array using a primer or probe specific to said DNA sequence; and (d) determining the function of said DNA sequence by comparing individuals comprising said mutation in said DNA sequence to corresponding individuals lacking said mutation in said DNA sequence. In the method, the DNA composition may comprise plant DNA. In particular embodiments the plant DNA may be further defined as monocot plant DNA, and may be still further defined as derived from a species selected from the group consisting of maize, rice, wheat, barley, sorghum, oat, and sugarcane. In particular embodiments, the monocot DNA comprises maize DNA. The plant DNA can also comprise dicot plant DNA, and may be still further defined as derived from a species selected from the group consisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. In particular embodiments, the DNA composition is Arabidopsis thaliana DNA.
Still yet another aspect of the invention provides a method for isolating a plant comprising a desired integration event. In particular embodiments of the invention, the method comprises the steps of: (a) integratively transforming a plurality of plants; (b) obtaining DNA from said plants; (c) amplifying a plurality of transgene insertion junctions from said DNA; (d) preparing at least a first array comprising said amplified insertion junctions; and (e) detecting a desired integration event with a probe or primer corresponding a preselected genomic region. In particular embodiments, the plant may be further defined as a monocot plant, and may be still further defined as derived from a species selected from the group consisting of maize, rice, wheat, barley, sorghum, oat, and sugarcane. In other embodiments, the monocot plant is a maize plant. The plant can also comprise a dicot plant, and may be still further defined as a species selected from the group consisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. In particular embodiments, the plant is an Arabidopsis thaliana plant.
Still yet another aspect of the invention provides a plant preparable by a process comprising the steps of: (a) integratively transforming a plurality of plants; (b) obtaining DNA from said plants; (c) amplifying a plurality of transgene insertion junctions from said DNA; (d) preparing at least a first array comprising said amplification insertion junctions; and (e) detecting a plant having a desired transgene insertion event using a probe or primer corresponding to the selected genomic region. The plant may be further defined as a monocot plant, wherein the monocot plant may be still further defined as a monocot plant selected from the group consisting of maize, rice, wheat, barley, sorghum, oat, and sugarcane. In particular embodiments the monocot plant is maize. The plant may also be a dicot plant, and in particular embodiments, still further defined as selected from the group consisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. In particular embodiments the dicot plant is an Arabidopsis thaliana plant.
The present invention represents a significant advance over prior methods for identifying insertional mutations in that it allows for the simultaneous screening of large numbers of unique insertion events. Therefore, the first step of the invention, in one embodiment, will involve obtaining or generating a population of individuals with insertional mutations from which to screen for the mutant of interest. A preferred population will represent a large number of insertional mutations such that there will be a high probability of identifying a mutant for any given locus within the population. In a preferred embodiment, the next step will generally involve isolating DNA from the population of insertional mutations and creating pools which contain DNA from various different combinations of individuals. The pools are designed such that, through analysis of multiple pools, sequences representing single members of a population can be identified without the need for individual analysis of each member of the population. The insertion junctions present in each pool are then amplified non-selectively, providing a broad class of xe2x80x9ctaggedxe2x80x9d insertion junctions which can subsequently be detected by use of gene-specific probes or primers. An efficient means employed for the detection of amplified insertion junctions in the pools is the preparation of arrays arranged on a suitable solid support material. The labeled gene-specific probes may then be hybridized and detected directly on the arrays, allowing simultaneous screening of a large number of pools and ultimate identification of one or more insertional mutants.
The probability of successfully identifying a chosen insertional mutant with the current invention will be greatly influenced by the characteristics of the starting population(s) from which insertional mutants will be screened. One important characteristic of the population will be the number of insertional mutations it contains. It will, of course, be preferred that any such population contain a sufficient number of insertion events that there is a reasonable likelihood of detecting at least one insertional mutant from any particular gene or locus. As such, the mechanism by which insertional mutations are generated will be important to the degree of ease with which the current invention may be used. While insertion mutations caused by potentially any known sequence long enough to be amplified may be detected with the current invention, certain types of insertions will offer advantages. Preferred insertion mutations will be predominately or completely randomly distributed throughout the target genome. This will decrease the likelihood that a particular locus is lacking an insertion mutation in the generated population and also reduce the size of the population needed to have a reasonable probability of detecting any given insertion mutation. Also preferred will be insertional mutagens which are capable of producing large numbers of mutations both within individuals and within populations, thereby increasing the effective number of mutations which may be obtained and subsequently screened. The insertion mutations created will also preferably alter gene expression for the mutated gene copy, allowing studies to elucidate the mutated genes"" phenotypic effect and function, and potentially creating valuable new phenotypes.
Examples of types of insertion mutations which are contemplated to be of particular utility with the current invention will be those created by transposable elements and transgenes introduced by transformation. Which type of these, or another, insertion mutations is utilized with the current invention will typically depend on factors including the organism being studied, available resources, and the goal of the study. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. In some monocot plants, however, transformation is less efficient and requires tissue culture steps which are comparatively time- and labor-intensive, making transformation a much less attractive alternative. Also, some species have lines with active transposable elements which can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. In particular instances, it may be advantageous to screen multiple types of insertion mutations, thereby increasing the chance of detecting any given desired mutant. Therefore, a number of factors will be taken into account when choosing the type(s) of insertion mutation to be identified with the current invention. These factors will be readily apparent to those of skill in the art in light of the present disclosure and will dependent on the specific goals of the investigation.
(i) Target Organism for Use with the Invention
The current invention is applicable to any species for which insertional mutants may be obtained. As such, it is specifically contemplated by the inventor that one may wish to use the current invention for the identification of specific insertion events from plants, animals, lower eukaryotes and prokaryotes. Examples of some animals for which the current invention may be used include poultry, dairy and beef cattle, primates, rodents, swine and insects. Examples of plants which are specifically contemplated for use with the current invention include monocots such as maize, rice, wheat, barley, sorghum, oat, and sugarcane, as well as dicots such as cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. Maize and Arabidopsis represent target plant species which will be particularly advantageous for use with the current invention.
(ii) Utilization of Transposon-Generated Insertion Mutations
Transposable-elements are an extremely versatile class of insertional mutagen in that a great variety of transposable elements have been identified, with representative elements having been found in all eukaryotic genomes examined (Flavell et al., 1992).
As used herein, the term xe2x80x9ctransposable elementxe2x80x9d will mean any mobile genetic element which is capable of replicative or non-replicative transposition within a genome, causing insertional mutagenesis at the site of insertion. One example of a transposable element of maize contemplated to have particular utility in the generation of insertion mutations is the Mutator element (Bennetzen, 1984; Talbert et al., 1989; see Genbank Accession Numbers: x14224, x14225, g22495, g22466, g22373, m76978 and x97569). Other examples of transposable elements which are deemed particularly useful insertional mutagens are the Ac element (Geiser et al., 1982; U.S. Pat. No. 4,732,856, specifically incorporated herein by reference in its entirety) and the tobacco element slide-124 (Grappin et al, 1996; Genbank Accession Number x97569).
(iii) Generation of Insertionally Mutagenized Plant Cells by Transformation
There are many methods for transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods are believed to include virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium infection (described in, for example, U.S. Pat. No. 5,591,616, specifically incorporated herein by reference in its entirety); direct delivery of DNA such as by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), by electroporation (U.S. Pat. No. 5,384,253), by agitation with silicon carbide fibers (Kaeppler et al. 1990), and by acceleration of DNA coated particles (U.S. Pat. No. 5,550,318), etc. Through the application of techniques such as these, certain cells from virtually any plant species may be stably transformed, and these cells developed into transgenic plants. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment and the like.
One type of insertional mutations which will be of particular use in the current invention are those caused by the T-DNA of Agrobacterium. An important advantage of T-DNA-based insertions is that they are apparently randomly distributed in any given genome (reviewed by Tinland, 1996). This has been confirmed in Arabidopsis, where a uniform distribution at the chromosomal level and a random distribution within translated and untranslated regions of genes was shown (Aspiroz-Leehan and Feldman, 1997). Moreover, sequence analysis of target sites shows that: (i) integration is not site-specific; (ii) T-DNA integration can lead to small deletions (13-72 bp) at the site of insertion; and (iii) the left-end border of integrated T-DNA is usually poorly conserved as compared to the right border sequences, which can be conserved up to the nucleotide that is covalently attached to the VirD2 movement protein (Tinland, 1996). Additionally, one or more T-DNA loci (chromosomal integration sites) can frequently be found integrated into the genome of a plant cell, and the same cell can carry T-DNAs derived from different Agrobacteria cells (DeBlock et al., 1991; Depicker, 1995). Frequently, the structure of the T-DNA at a locus can be complex, involving the integration of direct and inverted T-DNA repeats.
1. Electroporation
Where one wishes to introduce DNA by means of electroporation, it is contemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253, incorporated herein by reference in its entirety) will be particularly advantageous. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation, by mechanical wounding.
To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus, or alternatively one may transform immature embryos or other organized tissue directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Such cells would then be recipient to DNA transfer by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol, dependent on the nature of the newly incorporated DNA.
2. Microprojectile Bombardment
A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment (U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Patent Publication No. 94/09699; each specifically incorporated herein by reference in its entirety). In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. Hence, it is proposed that DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
An advantage of microprojectile bombardment, in addition to its being an effective means of reproducibly stably transforming monocots, is that neither the isolation of protoplasts (Christou et al., 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large. Examples of species for which the Biolistics Particle Delivery System has been successfully used for transformation include monocot species such as maize, barley, wheat, rice, and sorghum, as well as various dicot species, including tobacco, soybean, cotton, sunflower, and tomato.
For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens may be positioned between the acceleration device and the cells to be bombarded.
In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in the manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.
Accordingly, it is contemplated that one may wish to adjust various bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters. such as gap distance, flight distance, tissue distance, helium pressure, and microprojectile particle size. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration, and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. Results from such small scale optimization studies are disclosed herein, and the execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure (see, for example, PCT Patent Publication No. 94/09699, specifically incorporated herein by reference in its entirety).
3. Agrobacterium-Mediated Transfer
Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (Fraley et al., 1983; Rogers et al., 1987). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and the intervening DNA is usually inserted into the plant genome as described (Spielmann et al., 1986; Jorgensen et al., 1987).
Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987) have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer. An example of one T-DNA which will be especially useful with the current invention will be that of SEQ. ID NO. 1.
Agrobacterium-mediated transformation of leaf disks and other tissues, such as cotyledons and hypocotyls, appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants and is the preferable method for transformation of dicots, including Arabidopsis, tobacco, tomato, and potato. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors as described (Bytebier et al., 1987). Therefore, commercially important cereal grains, such as rice, corn, and wheat must usually be transformed using alternative methods. Agrobacterium-mediated transformation of maize and rice has, however, been described in U.S. Pat. No. 5,591,616, specifically incorporated herein by reference in its entirety.
One efficient means by which Agrobacterium plant transformation can be mediated is by way of vacuum infiltration. This procedure is based on the vacuum infiltration of a suspension of Agrobacterium cells containing a binary T-DNA vector into plant tissue, such as, for example, from Arabidopsis plants. Exemplary procedures for vacuum infiltration are known to those of skill in the art and are disclosed in Bechtold and Bouchez (1995); and Bechtold et al. (1993), each of which is specifically incorporated herein by reference in its entirety.
A transgenic plant formed using Agrobacterium transformation methods typically contains a single transgene or a few copies of a transgene on one chromosome. Such transgenic plants can be referred to as being hemizygous. For detection of an insertional mutagen, such a plant may be preferred, in that many of the mutations may be recessive lethals. Where the mutation is not a recessive lethal, a preferred plant may be homozygous for the added structural gene, i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a hemizygous transgenic plant that contains a single added gene, germinating some of the seed produced, and analyzing the resulting plants.
It is to be understood that two different transgenic plants can also be mated to produced offspring that contain multiple, independently-segregating added, insertion events. Specifically contemplated by the inventor, is the creation of plants which contain 1, 2, 3, 4, 5, or even more independently-segregating added insertion events. Selfing of appropriate progeny can produce plants that are homozygous for all added insertion mutations. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.
4. Other Transformation Methods
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).
Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimara et al., 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986; Omirulleh et al., and 1993 U.S. Pat. No. 5,508,184; each specifically incorporated herein by reference in its entirety).
To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, 1989). Also, pollen-mediated transformation may be used (U.S. Pat. No. 5,629,183; specifically incorporated herein by reference) In addition, xe2x80x9cparticle gunxe2x80x9d or high-velocity microprojectile technology can be utilized (Vasil, 1992).
Using that latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.
(iv) Generation of Insertionally Mutagenized Animal Cells by Transformation
In certain embodiments of the invention, animal cells comprising novel insertional mutants may be created by integrative transformation of recipient animal cells. Through such methods, which are well known to those of skill in the art, and others set forth herein, insertional mutants may be created for virtually any animal, plant, prokaryote or lower eukaryote. Specific methods contemplated by the inventor to be of use in the creation of insertional mutants are disclosed herein.
An example of a method of DNA delivery to recipient cells which may be used is viral infection, where a particular construct is encapsulated in an infectious viral particle. For use herein, the virus will be one which directs integrative transformation of the transformed cell. Non-viral methods for the transfer of foreign DNA into recipient cells also are contemplated in the present invention. In one embodiment of the present invention, the construct may consist only of naked DNA or plasmids; however, almost any DNA segment which is capable of insertionally mutating a target locus and which has a known sequence may potentially be used with the current invention. Transfer of the DNA may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane.
1. Liposome-Mediated Transfection
Foreign DNA may be delivered to cells by way of liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991). It is contemplated that one may wish to complex the DNA to be delivered with Lipofectamine (Gibco BRL).
Liposome-mediated nucleic acid delivery of foreign DNA in vitro has been demonstrated to be a reliable means of transformation (Nicolau et al., 1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
In certain embodiments, the liposomes may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
2. Electroporation
In certain embodiments of the present invention, insertionally mutagenized animal cells may be created via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. This technique is widely applicable to virtually any eukaryotic cell and may also be used for transformation of prokaryotes.
Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.
3. Calcium Phosphate Precipitation or DEAE-Dextran Treatment
In other embodiments of the present invention, the foreign DNA may be introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus DNA (Graham et al., 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3, and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
In another embodiment, the foreign DNA may be delivered into the cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleulemia cells (Gopal, 1985).
4. Direct Microinjection or Sonication Loading
In still further embodiments of the invention, insertionally mutagenized animal cells may be created by the delivery of foreign DNA with microinjection or sonication loading. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985), and LTKxe2x88x92 fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987). A similar method involves injecting a polyamino acid/DNA complex into the cytoplasm of animal cells to effect transformation (U.S. Pat. No. 5,523,222 specifically incorporated herein by reference).
5. Receptor-Mediated Transfection
A still further method for delivery of foreign DNA involves the delivery of constructs to the target cells with receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the transformation. Specific delivery in the context of another mammalian cell type is described by Wu and Wu (1993).
Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a DNA-binding agent. Others comprise a cell receptor-specific ligand to which the DNA construct to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; European Patent No. 0 273 085), which establishes the operability of the technique. In the context of the present invention, the ligand will be chosen to correspond to a receptor specifically expressed on the neuroendocrine target cell population.
In other embodiments, the DNA delivery vehicle component of a cell-specific gene targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acids to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptors of the target cell and deliver the contents to the cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.
In still further embodiments, the DNA delivery vehicle component of the targeted delivery vehicles may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
Therefore, transformation of host species may be used in a similar manner to transposon-tagging. In transposon tagging, as with integrative transformation, insertion mutations are created in the genomes of target organisms by transposable elements. This creates mutant individuals from which mutant phenotypes can be identified. DNA can then be isolated from the mutants and used for the creation of genomic libraries. The mutated gene can then be efficiently cloned through the use the transposon as a xe2x80x9ctagxe2x80x9d. Typically, a number of candidate genes will first be identified. These may then be confirmed by complementation experiments or DNA sequencing and homology searches for related known genes.
I. Amplification of Insertion Junctions
An important aspect of the current invention is that it allows selection of specific insertional mutants from a diverse class of insertion events. For this purpose, one step of the invention utilizes the non-selective amplification of insertion junctions. As used herein, the term xe2x80x9cnon-selective amplificationxe2x80x9d is used to denote amplification procedures which will simultaneously amplify a broad class of insertion junctions without the need for a single gene-specific primer. Techniques which are contemplated by the inventor as being particularly useful for the non-specific amplification are inverse PCR, vectorette PCR, and primer-adapted PCR, with vectorette PCR being most preferred, although potentially any method capable of amplifying a diverse class of insertion junctions may be used.
(i) Inverse PCR
Inverse polymerase chain reaction (IPCR) is an extension of the polymerase chain reaction that permits the amplification of regions that flank any DNA segment of known sequence, either upstream or downstream (see U.S. Pat. No. 4,994,370, specifically incorporated herein by reference in its entirety). The essence of IPCR is that, by circularizing a restriction enzyme fragment containing a region of known sequence plus flanking DNA, PCR can be performed using oligonucleotides whose sequence is taken from the single region of known sequence and oriented with respect to one another such that their 5xe2x80x2 to 3xe2x80x2 extension products proceed toward each other by going xe2x80x9caround the circlexe2x80x9d through what originally was flanking DNA. This leads to the amplification of DNA strands containing what was originally flanking DNA. The advantage of a technique such as IPCR, with respect to the current invention, is that using a single primer set one may amplify a representative sample of insertion junctions from a particular group of individuals.
Selection of appropriate restriction enzymes for use in IPCR can be determined empirically by Southern blotting and hybridization procedures using all or part of the core region. Selection of the appropriate fragment can be facilitated by computer search methods, since in most cases the entire nucleotide sequence of the core (e.g., well characterized insertional mutagens such as transposable elements or transgenes) region will be known. The amplified fragment should be no greater than 2-3 kilobases (kb), which is a limitation imposed by the size of a region that can be efficiently amplified using the most commonly available methods of PCR. However, recently PCR techniques have been developed, termed Long PCR, which are capable of amplifying DNA fragments of 20 kb or more.
After restriction enzyme digestion, the DNA fragments produced by the restriction enzyme are diluted and ligated under conditions that favor the formation of monomeric circles (Collins et al., 1984). The resulting intramolecular ligation products are then used as substrates for enzymatic amplification by PCR using oligonucleotide primers homologous to the ends of the core sequence but facing in opposite orientations. The primary product of the resulting amplification is a linear double-stranded molecule including segments situated both 5xe2x80x2 and 3xe2x80x2 to the core region. The junction between the original upstream and downstream regions, otherwise ambiguous, can be identified as the restriction site of the restriction enzyme that was used to produce the linear fragments prior to ligation. By selecting a restriction enzyme that cleaves inside a known core sequence, the IPCR procedure will produce products containing only the upstream or only the downstream flanking regions.
(ii) Vectorette PCR
There are three basic steps in the technique of vectorette PCR: (1) digestion of target DNA with one or more suitable restriction enzymes; (2) ligation of suitable synthetic oligonucleotides onto the digested DNA; and (3) PCR using a specific primer and a primer directed toward the synthetic oligonucleotides (see European Patent No. 0 439 330, specifically incorporated herein by reference in its entirety). In this procedure, nonspecific amplification of all digested fragments is avoided by the design of specific fragments of synthetic DNA, called vectorettes. Vectorettes are designed so that they can be amplified only if they are attached to the DNA insertional mutagen. The vectorette is only partially double-stranded and contains a central mismatched region. The vectorette PCR primer has the same sequence as the bottom strand of this mismatched region and therefore has no complementary sequence to anneal to in the first cycle of PCR. In the first cycle of PCR, only the known primer, which is directed toward the insertional mutagen, will prime DNA synthesis. This will produce a complementary strand for the vectorette PCR primer to anneal to in the second cycle of PCR. In the second and subsequent cycles of PCR, both primers can prime synthesis, with the end result being that the only fragment amplified contains the insertional mutagen and flanking DNA of the insertion site.
(iii) Primer Adapted PCR
The primer-adapted PCR technique is a derivation of ligation-mediated single-sided PCR (Fors et al., 1990; Mueller et al., 1989). This method uses linker ligation and subsequent amplifications with a linker-primer and multiple insertional-mutagen-specific primers (xe2x80x9cnestedxe2x80x9d primers) to obtain specificity. The ligation-mediated single-sided PCR protocol involves multiple PCRs and subsequent purifications on agarose gels.
The amplification procedure involves, as a first step, restriction with an appropriate restriction enzyme, such as Sau3AI, and ligation of primer adapters to the different DNA size fractions. Then, approximately 50 cycles of linear amplification are performed using an internal biotinylated primer complimentary to the insertional mutagen. The biotinylated linear PCR product is purified from the rest of the genomic DNA with streptavidin-coated magnetic beads and subjected to exponential PCR using the adapter-primer and the insertional-mutagen specific primer. The result of this first round of exponential PCR may be visualized on an agarose gel and used in the preparation of arrays. Successful, specific amplification should be indicated by a series of bands on the agarose gel.
In order to avoid the purification steps required because of non-specificity in the PCR, an additional step may be introduced that involves linear amplification of the target sequence with a biotinylated primer and separation of the product with the aid of streptavidin-coated magnetic beads (Hultman et al., 1989; Rosenthal and Jones, 1990). This strategy may be employed in combination with ligation of oligo-cassettes to restricted DNA to directly amplify unknown regions which flank an insertional mutagen (Rosenthal and Jones, 1990).
The basic concept of the method is to employ an xe2x80x9cinternalxe2x80x9d primer complementary to a known sequence in the insertional mutagen in combination with an xe2x80x9cexternalxe2x80x9d adapter-primer. First, primer adapters are ligated onto the genomic DNA digested with a suitable enzyme (for example, Sau3AI), then a linear PCR is performed with the insertional mutagen-complimentary primer, which is biotinylated. Since the linear PCR product is biotinylated, it can then be purified from the rest of the genomic DNA with the aid of streptavidin-coated magnetic beads. After the magnetic purification, an exponential PCR is carried out using the internal primer in combination with the adapter-primer. An extra round of PCR with a nested internal primer and the adapter-primer can be performed to achieve increased specificity. The amplified product can then be used for the production of arrays for ultimate detection of insertional mutants.
(iv) Other Methods
As previously stated, any method which may be used to enrich for a diverse collection of insertion junctions may be used with the current invention. An example of one such technique disclosed herein for the enrichment of transposon Mu-tagged sites is Amplification of Insertion Mutagenized Sites (AIMS), the procedure for which is outlined below, in Example 5 and described by Souer et al. 1995.
II. Detection of Insertional Mutants from Arrays
One aspect of the current invention which allows for efficient selection of large numbers of insertional mutants is the creation of arrays comprising insertion-junction-enriched DNA pools. The precise placement of this pooled DNA into specific arrays allows for the simultaneous screening of potentially thousands of insertion mutations. The method involves the placement and binding of DNA to known locations, termed sectors, on a solid support. Through hybridization of a desired specific probe or primer to the array, for example, insertion mutations corresponding to that gene may be identified from the total collection of insertional mutants. Further, because the amplification step may be conducted repeatedly, a large number of identical or non-identical arrays may be produced, thereby allowing simultaneous screening with many different locus-specific probes or primers.
Many different methods for preparation of arrays of DNA on solid supports are known to those of skill in the art. Specific methods of which are disclosed in, for example, Affinity Techniques, Enzyme Purification: Part B, Meth. Enz. 34 (ed. W. B. Jakoby and M. Wilchek, Acad. Press, N.Y. (1974) and Immobilized Biochemicals and Affinity Chromatography, Adv. Exp. Med. Biol. 42 (ed. R. Dunlap, Plenum Press, N.F. 1974), each specifically incorporated herein by reference in its entirety). Examples of other techniques which have been described include the use of successive application of multiple layers of biotin, avidin, and extenders (U.S. Pat. No. 4,282,287, specifically incorporated herein by reference in its entirety); through methods employing a photochemically active reagent and a. coupling agent which attaches the photoreagent to the substrate (U.S. Pat. No. 4,542,102, specifically incorporated herein by reference in its entirety), use of polyacrylamide supports on which are immobilized oligonucleotides (PCT Patent Publication No. 90/07582, specifically incorporated herein by reference in its entirety), through use of solid supports on which oligonucleotides are immobilized via a 5xe2x80x2-dithio linkage (PCT Patent Publication No. 91/00868, specifically incorporated herein by reference in its entirety); and through use of a photoactivateable derivative of biotin as the agent for immobilizing a biological polymer of interest onto a solid support (see U.S. Pat. No. 5,252,743; and PCT Patent Publication No. 91/07087 to Barrett et al., each specifically incorporated herein by reference in its entirety). In the case of a solid support made of nitrocellulose or the like, standard techniques for UV-crosslinking may be of particular utility (Sambrook et al., 1989).
The solid support surface upon which the array is produced may potentially be any suitable substance. Examples of materials which may be used include polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, etc. It may also be advantageous to use a surface which is optically transparent, such as flat glass or a thin layer of single-crystal silicon. Contemplated as being especially useful are nylon filters, such as Hybond N+ (Amersham Corporation, Amersham, UK). Surfaces on the solid substrate will usually, though not always, be composed of the same material as the substrate, and the surface may further contain reactive groups, which could be carboxyl, amino, hydroxyl, or the like.
It is contemplated that one may wish to use a surface which is provided with a layer of crosslinking groups (U.S. Pat. No. 5,412,087, specifically incorporated herein by reference in its entirety). Crosslinking groups could be selected from any suitable class of compounds, for example, aryl acetylenes, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, diacids, amino acids, or combinations thereof. Crosslinking groups can be attached to the surface by a variety of methods that will be readily apparent to one of skill in the art. For example, crosslinking groups may be attached to the surface by siloxane bonds formed via reactions of crosslinking groups bearing trichlorosilyl or trisalkoxy groups with hydroxyl groups on the surface of the substrate. The crosslinking groups can be attached in an ordered array, i.e., as parts of the head groups in a polymerized Langmuir Blodgett film. The linking groups may be attached by a variety of methods that are readily apparent to one skilled in the art, for instance, by esterification or amidation reactions of an activated ester of the linking group with a reactive hydroxyl or amine on the free end of the crosslinking group.
The ultimate goal of producing an array in accordance with current invention, will be in screening large numbers of individuals or subsets of individuals for detection of an insertional mutant. Therefore, once the array is produced, the first step will, in a preferred embodiment, involve hybridizing the array with a solution containing a marked (labeled) probe. For detection of a mutation in a specific gene, this will typically involve the use of a cloned DNA segment including that gene sequence as a probe. Following hybridization, the surface is then washed free of unbound probe, and the signal corresponding to the probe label is identified for those regions on the surface where the probe has high affinity. Suitable labels for the probe include, but are not limited to, radiolabels, chromophores, fluorophores, chemiluminescent moieties, antigens and transition metals. In the case of a fluorescent label, detection can be accomplished with a charge-coupled device (CCD), fluorescence microscopy, or laser scanning (U.S. Pat. No. 5,445,934, specifically incorporated herein by reference in its entirety). When autoradiography is the detection method used, the marker is a radioactive label, such as 32P, and the surface is exposed to X-ray film, which is developed and read out on a scanner or, alternatively, simply scored manually. With radiolabeled probes, exposure time will typically range from one hour to several days. Fluorescence detection using a fluorophore label, such as fluorescein, attached to the ligand will usually require shorter exposure times. Alternatively, the presence of a bound probe may be detected using a variety of other techniques, such as an assay with a labeled enzyme, antibody, or the like. Other techniques using various marker systems for detecting bound ligand will also be readily apparent to those skilled in the art.
Detection may, alternatively, be carried out using PCR. In this instance, PCR detection may be carried out in situ on the slide. In this case one may wish to utilize one or more labeled nucleotides in the PCR mix to produce a detectable signal. Detection may also be carried out in a standard PCR reaction on the prepared samples to be screened. For this type of detection, the sectors in the array will not consist of DNA bound to solid support but will consist of DNA samples in solution in the wells of a microtiter dish.
It also is contemplated by the inventor that one may xe2x80x9creversexe2x80x9d the above described detection protocols. For example, instead of using amplified insertion junctions for preparation of a detectable array, one could use genetic sequences which are specific to the locus for which an insertion mutation is desired. In this case, one could label the amplified insertion junctions and use then as probes for the detection of loci corresponding to the insertion mutation. Therefore, by multiple hybridizations with different pools of amplified insertion junctions, one may ultimately identify individuals having the desired insertion mutations.
As an alternative to detection of insertion junctions with PCR or hybridizations, sequencing of insertion junctions may be used. In this procedure one would preferably first prepare pools of DNA from individuals having insertion junctions. The pools may be designed such the source of a particular insertion junction can be identified without the need for screening of all individuals within a population. An exemplary pooling procedure comprises the designation of individuals into a 2xc3x972 grid. Pools of DNA are then prepared from all of the individuals within each column and row. The identification of a sequence in a column and a row will thereby provide a precise coordinate for the individual having that sequence. Alternatively, pools needn""t be used, however, this will be less preferred as more effort will be needed to find a specific desired insertion.
III. Competitive Hybridizations
Use of the current invention may, in particular circumstances, require competitive hybridizations. This may be so when the locus-specific probe used contains one or more sequences which are repeated throughout the target genome, thereby leading to detection of multiple, non-specific loci. The situation will arise more frequently where probes are derived from genomic DNA clones of organisms which have relatively large genomes such as many mammals, and particularly plants such as maize and wheat.
Signal from repetitive sequences may be xe2x80x9cblockedxe2x80x9d by inclusion of unlabeled total genomic DNA in the mixture of labeled probe DNA, or by use of the unlabeled DNA in prehybridizations before application of the labeled probe. Even more effective than total genomic DNA for blocking will be DNA which is xe2x80x9cenrichedxe2x80x9d for repetitive, such as Cot-1 DNA (Zwick et al., 1997, specifically incorporated herein by reference in its entirety). It is also contemplated that one may wish to use blocking DNA which contains unlabeled sequences of the insertional mutagen. This may help to avoid detection of the insertional mutagen and help ensure only detection of the flanking sequences.
The proportion of blocking DNA to probe DNA used will vary and will depend on a number of variables. Factors upon which the concentration used is dependent include: the relative proportion of repetitive sequences in the probe/primer and target sequences, the desired level of sensitivity in the detection, the size of the repetitive sequences, and the degree of sequence homology between the probe repetitive sequences and those of the target. Typical concentrations of unlabeled blocking DNA which may be used include from about 20 to about 200 fold excess, relative to the probe, including about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, and 190 fold excess, Alternatively, one may wish to use concentrations of blocking DNA greater or lesser than this range, including about 10, 300, 400, 500, 600, 700, 800, 900, or about 1000 fold excess. The optimal concentration used, however, will be dependent on the above mentioned factors and will be known to those of skill in the art in light of the present disclosure. It is noted, however, that while competitive hybridizations are effective in eliminating background signal caused by repetitive sequences, it will be preferable to avoid the problem through use of unique or low copy probe sequences, such as, for example, cDNAs.
IV. Use of the Invention for Discovery of Gene Function
An important use of the current invention will be in acquiring information regarding the function of genes. Therefore, one embodiment of the invention involves the identification and isolation of a mutant for a selected gene and the use of that mutant in studies of gene function. By comparison of the phenotype of one or more individuals having a particular insertion mutation to a representative sample of individual without the mutation, inferences may be made regarding the function of the mutated sequence.
In this manner, one may begin with a cDNA or other probe or primer specific for a genetic sequence of unknown function, and, through use of the current invention, obtain information regarding the function of that sequence. In light of the high-throughput-capability of the current invention, one could, alternatively, systematically obtain large numbers of mutants and screen the mutants for identification of genes associated with traits of interest. For example, one may use a sample of plant cDNA probes to isolate maize plants having mutations corresponding the cDNAs. These mutants may then be grown in the field and various observations made of the mutant phenotype including characteristics such as yield, disease or pest resistance, stress tolerance, or any other trait deemed of interest. A correlation between a particular mutant and a phenotype will, of course, suggest that the mutated gene is involved in the expression of that trait. The mutated gene can then be cloned or used for further studies as desired by the user of the invention. Such studies may involve, for example, operably linking the cloned gene to a different promoter and using the construct created to transform plants.
V. Expression Analysis
Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA will only be expressed in particular cells or tissue types, and hence it will be necessary to prepare RNA for analysis from these tissues. PCR techniques may also be used for detection and quantitation of RNA produced from introduced genes. In the application of PCR, it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then, through the use of conventional PCR techniques amplify the DNA. In most instances, PCR techniques, while useful, will not demonstrate the integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.
While Southern blotting and PCR may be used to detect the gene(s) in question, they do not provide information as to whether the gene is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.
Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques, such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity, such as western blotting, in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest, such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
Assay procedures also may be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and 14C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.
Very frequently, the expression of a particular mutant is determined by evaluating the phenotypic results of its expression. These assays also may take many forms, including, but not limited, to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins which change amino acid composition and may be detected by amino acid analysis, or by enzymes which change starch quantity, which may be analyzed by near infrared reflectance spectrometry.
VI. Genetic Characterization of Insertional Mutants
To confirm the presence of one or more insertional mutants in an individual, to track these in progeny, and to analyze the effects of a particular mutation, a variety of assays may be performed. Such assays include, for example, xe2x80x9cmolecular biologicalxe2x80x9d assays, such as Southern and Northern blotting and PCR; xe2x80x9cbiochemicalxe2x80x9d assays, such as detecting the presence or absence of a particular protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also by analyzing the phenotype of the whole regenerated plant.
(i) DNA Integration, RNA Expression and Inheritance
Genomic DNA may be isolated from any plant or animal cells to determine the presence of a particular insertional event using techniques well known to those skilled in the art. The presence of an insertional mutant may, for example, be determined by polymerase chain reaction (PCR). Using this technique, discrete fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis will permit one to follow a particular insertional mutant in the offspring of a cross. Insertional mutants are expected to be generated randomly and, for this reason, are expected to be unique, based on their genomic location. Thus, by designing PCR primers which will amplify segments which include both the inserting DNA and the subsequently mutated native sequence, unique amplification products which are specific to that insertion event can be identified.
Southern hybridization is especially useful for identification of particular insertional mutants, in that each insertional mutant is expected to have a unique restriction pattern. Using this technique specific, DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence, the Southern hybridization pattern of a given insertion event serves as an identifying characteristic of that transformant. The technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence of an integration event, but also characterizes each individual insertion event.
Both PCR and Southern hybridization techniques can be used to demonstrate transmission of an insertional mutant to progeny. In most instances, the characteristic Southern hybridization pattern for a given insertional mutation will segregate in progeny as one or more Mendelian genes (Spencer et al., 1992), indicating stable inheritance of the transgene.
For use as a probe, one may use DNA of the insertional mutagen, from the mutated endogenous sequence, or from both. In the case of an insertional mutagen which is present in low copy, it may be desirable to use DNA from the insertional mutagen as a probe. However, where the insertional mutagen is present in high copy, such as will be the case with endogenous transposable elements, the detected restriction patterns will be complex and difficult to interpret. In this case, it may be desirable to use the endogenous, mutated sequence as a probe.
The biological sample for assays may potentially be any type of plant or animal tissue. Nucleic acid may be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA; in another, it is poly-A RNA. Normally, the nucleic acid is amplified.
Depending on the format, the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e.g.. ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of a radiolabel or fluorescent label, or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
Following detection, one may compare the results seen in a given mutant with a statistically significant reference group of non-mutated controls. Typically, the non-mutated control will be of a genetic background similar to the mutated individual. In this way, it is possible to detect differences in the amount or kind of protein detected in various different mutants.
A variety of different assays are contemplated in the screening of insertional mutants isolated using the methods of the current invention. These techniques can be used to detect for both the presence of particular mutations as well as the resulting effects caused by the mutations. The techniques include, but are not limited to, fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern or Northern blotting, single-stranded conformation analysis (SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, denaturing gradient gel electrophoresis, RFLP, and PCR-SSCP.
(ii) Primers, Probes and Template-Dependent Amplifications
The term primer, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from 10 to 20 base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred. Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to bind to the target DNA or RNA and need not be used in an amplification process. In preferred embodiments, the probes or primers are labeled with radioactive species (32P, 14C, 35S, 3H, or other label), with a fluorophore (rhodamine, fluorescein), an antigen (biotin, streptavidin, digoxigenin), or a chemiluminescent (luciferase).
A number of template-dependent processes are available to amplify the sequences present in a given sample. One of the best known amplification methods is the polymerase chain reaction which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, each specifically incorporated herein by reference in its entirety.
Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the template to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described by Sambrook et al. (1989). Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases and are described in WO 90/07641, filed Dec. 21, 1990.
Another method for amplification is the ligase chain reaction (xe2x80x9cLCRxe2x80x9d), disclosed in European Patent No. 0 320 308, specifically incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and, in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as xe2x80x9ctarget sequencesxe2x80x9d for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
Qbeta Replicase, described in PCT Patent Publication No. PCT/US87/00880, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.
An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5xe2x80x2-[alpha-thio]-triphosphates in one strand of a restriction site, may also be useful in the amplification of nucleic acids in the present invention (Walker et al., 1992).
Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3xe2x80x2 and 5xe2x80x2 sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe are identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe, and the reaction is repeated.
Still another amplification method, described in GB Application No. 2 202 328 and in PCT Patent Publication No. PCT/US89/01025 (each specifically incorporated herein by reference in its entirety), may be used in accordance with the present invention. In the former application, xe2x80x9cmodifiedxe2x80x9d primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes is added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al.; PCT Patent Publication No. WO 88/10315; each specifically incorporated herein by reference in its entirety). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double-stranded DNA molecules are heat denatured again. In either case, the single-stranded DNA is made fully double-stranded by the addition of a second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase, such as T7 or SP6. In an isothermal cyclic reaction, the RNA""s are reverse transcribed into single-stranded DNA, which is then converted to double-stranded DNA, and then transcribed once again with an RNA polymerase, such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.
European Patent Application No. 0 329 822 (specifically incorporated herein by reference in its entirety) discloses a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (xe2x80x9cssRNAxe2x80x9d), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5xe2x80x2 to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large xe2x80x9cKlenowxe2x80x9d fragment of E. coli DNA polymerase 1), resulting in a double-stranded DNA (xe2x80x9cdsDNAxe2x80x9d) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle, leading to very swift amplification. With the proper choice of enzymes, this amplification can be done isothermally without the addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
PCT Patent Publication No. WO 89/06700 (specifically incorporated herein by reference in its entirety) discloses a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (xe2x80x9cssDNAxe2x80x9d) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include xe2x80x9cRACExe2x80x9d and xe2x80x9cone-sided PCRxe2x80x9d (Frohman, 1990; Ohara et al., 1989; each specifically incorporated herein by reference in its entirety).
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting xe2x80x9cdi-oligonucleotide,xe2x80x9d thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention (Wu et al., 1989, specifically incorporated herein by reference in its entirety).
(iii) Detection Methods
Products may be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to X-ray film or visualized under the appropriate stimulating spectra, following separation.
In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
In one embodiment, detection is by a labeled probe. The techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols (see Sambrook et al., 1989). For example, chromophore or radiolabeled probes or primers identify the target during or following amplification.
One example of the foregoing is described in U.S. Pat. No. 5,279,721 (specifically incorporated herein by reference in its entirety), which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
In addition, the amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques. Within certain methods, exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optima sequencing (Pignon et al., 1994). The present invention provides methods by which any or all of these types of analysis may be used.
(iv) Design and Theoretical Considerations for Relative Quantitative RT-PCR.
Reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR) can be used to determine the relative concentrations of specific mRNA species isolated from plants. By determining that the concentration of a specific mRNA species varies, it is shown that the gene encoding the specific mRNA species is differentially expressed.
In PCR, the number of molecules of the amplified target DNA increases by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles. If a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point, the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
The concentration of the target DNA in the linear portion of the PCR amplification is directly proportional to the starting concentration of the target before the reaction began. By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is only true in the linear range of the PCR reaction.
The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCR for a collection of RNA populations is that the concentrations of the amplified PCR products must be sampled when the PCR reactions are in the linear portion of their curves.
The second condition that must be met for an RT-PCR experiment to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard. The goal of an RT-PCR experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
Most protocols for competitive PCR utilize internal PCR standards that are approximately as abundant as the target. These strategies are effective if the products of the PCR amplifications are sampled during their linear phases. If the products are sampled when the reactions are approaching the plateau phase, then the less abundant product becomes relatively over-represented. Comparisons of relative abundances made for many different RNA samples, such as is the case when examining RNA samples for differential expression, become distorted in such a way as to make differences in relative abundances of RNAs appear less than they actually are. This is not a significant problem if the internal standard is much more abundant than the target. If the internal standard is more abundant than the target, then direct linear comparisons can be made between RNA samples.
The above discussion describes theoretical considerations for an RT-PCR assay for plant tissue. The problem inherent in plant tissue samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target). Both of these problems are overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5 to 100-fold higher than the mRNA encoding the target. This assay measures relative abundance, not absolute abundance of the respective mRNA species.
Other studies may be performed using a more conventional relative quantitative RT-PCR assay with an external standard protocol. These assays sample the PCR products in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling must be empirically determined for each target cDNA fragment. In addition, the reverse transcriptase products of each RNA population isolated from the various tissue samples must be carefully normalized for equal concentrations of amplifiable cDNAs. This consideration is very important since the assay measures absolute mRNA abundance. Absolute mRNA abundance can be used as a measure of differential gene expression only in normalized samples. While empirical determination of the linear range of the amplification curve and normalization of cDNA preparations are tedious and time consuming processes, the resulting RT-PCR assays can be superior to those derived from the relative quantitative RT-PCR assay with an internal standard.
One reason for this advantage is that, without the internal standard/competitor, all of the reagents can be converted into a single PCR product in the linear range of the amplification curve, thus increasing the sensitivity of the assay. Another reason is that, with only one PCR product, display of the product on an electrophoretic gel or another display method becomes less complex, has less background, and is easier to interpret.
(v) Chip Technologies
Specifically contemplated by the present inventor are chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization (see also, Pease et al., 1994; and Fodor et al., 1989).
VII. Definitions
Corresponding Individual Lacking an Insertion Mutation: an individual which has the same genetic background as another individual, but differs on the basis of lacking a particular insertion mutation.
Detectable Array: an arrangement of nucleic acid sequences from which specific sequences or subsets of sequences can be identified. The array can comprise DNA sequences bound to a solid support and can also include DNA compositions arranged in solution in suitable containers. For the purposes of the current invention the sequences will be ones which may be used to identify one or more specific insertion junctions. These sequences can, therefore, represent DNA of insertion junctions or, alternatively, sequences representing a particular locus for which an insertion mutation is desired.
DNA Composition Enhanced for a Plurality of Insertion Junctions: a DNA composition in which a non-locus specific selection of insertion junctions has been enhanced relative to the starting DNA from which the DNA composition is derived. Such non-locus specific selections are prepared without the need for use of probes or primers which are specific to the locus or loci for which an insertion mutation is desired. The selection procedure will typically, instead, use probes or primers which are specific to the insertional mutagen. Examples of such procedures include inverse PCR, primer adapted PCR, and vectorette PCR, AIMS, or any other amplification or isolation procedure which is capable of being used to enhance a DNA composition for a diverse class of insertion junctions.
Hybridization Filter: an object to which nucleic acids can be fixedly attached, and to which probes may be hybridized, for example, in Southern Hybridization. Exemplary hybridization filters will be made of nitrocellulose or nylon, although any similar materials may also be used.
Insertion Junction: the segment of DNA encompassing the end of an insertional mutagen and particularly, the flanking genomic DNA into the insertional mutagen has inserted. For the purposes of the invention, DNA from the insertional mutagen itself need not typically be present, but for detection, the flanking genomic DNA should be.
Insertional Mutagen: any sequence which is capable of inserting into a segment of genomic DNA thereby causing an insertion mutation.
Microscope Slide: an object similar to a standard slide used for holding a specimen to be observed under a microscope. The microscope slide will preferably be made of glass or a similar material and will have a flat surface, however, it will be understood to those of skill in the art that various trivial modifications may be made to a typical microscope slide and still not depart from the scope and meaning of the term as defined in the current invention.
Pool: a composition of DNA made from the combination of DNA from multiple individuals. The pool will typically be constructed to allow the identification of individuals possessing a desired genetic sequence from a populations of individuals without necessitating screening of every individual within that population.
VIII. Examples
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.